Oligopeptide lyophilisate, their preparation and use

ABSTRACT

A novel lyophilizate and method of preparation as well as the use of the lyophilizate to treat female infertility and for gonad protection. Cetrorelix is dissolved in acetic acid 30% v/v, the solution is transferred to water and freeze dried.

[0001] This is a continuation-in-part application of U.S. Ser. No.08/198,037, filed Feb. 22, 1994.

[0002] The present invention relates to the preparation of alyophilizate of a peptide and the use of the lyophilizate in thetreatment of infertility and to provide male gonad protection.

BACKGROUND OF THE INVENTION

[0003] Cetrorelix is a decapeptide with a terminal acid amide group thatis used in the form of its acetate salt. The synthesis and somepharmacological effects are described in European patent application 299402 (U.S. Pat. No. 4,800,191).

[0004] It should be possible to administer the active substancesubcutaneously in a dose of 0.1 to 20 mg. Aqueous solutions of thedecapeptide are unstable, and, therefore, autoclaving in the containerused to distribute it is not possible. During conventionalsterilization, using the prescribed conditions, the decapeptide tends todecompose. To obtain an injectable solution it was therefore necessaryto develop a lyophilizate.

[0005] The amount of active substance in the solution to be lyophilizedis, however, so small that, in low active substance concentrations, onlya loose fluff results on the glass wall of the ampoule after drying thesolution free of auxiliary substances, and this fluff is carried out ofthe vial with the stream of water vapor generated by the sublimationprocess. It is therefore necessary to use a bulking agent that forms astable cake. In high concentrations, this auxiliary substance can bedispensed with. The following auxiliary substances may be considered asbulking agents: hexitols, in particular mannitol, glucitol, sorbitol,such as D-sorbitol, dulcitol, allitol, altritol (for example D- andL-altritol), iditol (for example D- and L- iditol), their opticallyactive forms (D- and L-forms) as well as the corresponding racemates.Nannitol is used in particular, such as D-mannitol, L-mannitol,DL-mannitol, sorbitol and/or dulcitol, and, of these, D-mannitol ispreferred. The hexitol used may also be composed of a mixture of thehexitols named, for example a mixture of mannitol and sorbitol and/ordulcitol. Since dulcitol is less water soluble than, for example,mannitol, the dulcitol content in the aqueous solution should notexceed, for example, 3 percent by weight. Mannitol and sorbitol, on theother hand, can for example be mixed in any ratio.

[0006] Apart from hexitol it is also possible to add other, conventionalpharmaceutical auxiliary substances, such as amino acids, such asalanine, glycine, lysine, phenylalanine, asparaginic acid, glutaminicacid, leucine, lactose, polyvinylpyrrolidone, glucose, fructose, albuminand equivalent bulking agents. Urea and sodium chloride may also be usedas bulking agents. The total amount of such substances in the solutionwhich is used for freeze-drying, is for example 0-16.9 parts by weight,for example 0.1-7 parts by weight, based on 1 part by weight ofcetrorelix. In the finished lyophilizate the total amount of suchauxiliary substances may be up to 16.9 parts by weight, based on onepart by weight of hexitol. In detail, the amount of such auxiliarysubstances depends on the amount of hexitol present and to such anextent that the total amount of hexitol and such other auxiliarysubstances in the finished lyophilizate may not be more than a maximumof 17 parts by weight, based on 1 part by weight of cetrorelix. If only0.1 part by weight of hexitol is present in the lyophilizate, it is thuspossible to have up to 16.9 parts by weight of other auxiliarysubstances; if, for example, 8.5 parts by weight of hexitol are present,the amount of other auxiliary substances may for example be up to 8.5parts by weight, based on 1 part by weight of cetrorelix.

[0007] It was, however, found, during development work on thelyophilizate, that the active substance behaves in a widely variable andunpredictable manner during processing. The first batches gave goodresults, but it soon transpired that difficulties occurred duringsterile filtration and faulty batches resulted.

[0008] It is known from the literature, for example from Powell, M. F.;Pharmaceutical Research, 1258-1263 (8)1991; Dathe, M: Int. J. PeptideProtein Res. 344-349 (36) 1990; Szejtli, J.: Pharmaceutical TechnologyInternational 16-22, 1991 that oligopeptides, particularly those withterminal acid amide function, tend to form gels. During sterilefiltration this is apparent from the speed of filtration, indeed theincreased viscosity of such solutions can often already be detectedorganoleptically. A gelatinous layer remains on the sterile filter. Itis then no longer possible to prepare a medication with an exactly andreproducibly defined active substance content.

[0009] Table 1 lists various results of the first 11 batches.

[0010] The active substance contents fluctuate between 100% and 36%.TABLE 1 Cetrorelix acetate Batch Dosage Active substance content % 1 100μg 100 2 500 μg 100 3 500 μg  90 4 500 μg  36 5 500 μg 100 6 500 μg  857 1 mg  80 8 1 mg 100 9 2 mg 100 10 2 mg  80 11 2 mg 100

[0011] To avoid this gel formation, the literature lists the followingadditives which may be tried out on an experimental basis:

[0012] Organic solvents may be considered, for example acetonitrile,n-butanol, tertiary butanol, ethanol, isopropanol, octanol and benzylalcohol. It is also possible to use salts and buffer solutions, such asacetate buffer, citrate buffer, sodium chloride, sodium phosphate,sodium EDTA, sodium bicarbonate, phosphate buffer, guanidine acetate,urea.

[0013] Polymers may also be used, such as gelatin, polyethylene glycol600, hydroxyethyl starch, polyvinylpyrrolidone, polyvinyl alcohol. Theuse of amino acids, for example alanine, glycine, lysine, phenylalanine,asparaginic acid, glutaminic acid and leucine has also been described.Acids that were used were citric acid, caprylic acid, octanoic acid,hydrochloric acid, sulphuric acid and acetic acid. Physiologicallyacceptable surfactants that may be used are benzalkonium chloride, cetylalcohol, bile acids, lecithins, polysorbates, Spans® and Pluronics®.

[0014] Carbohydrates and cyclodextrins such as glucose, lactose,mannitol, saccharose, alpha-, beta- and gamma cyclodextrins,hydroxypropyl-alpha- and beta-cyclodextrins, hydroxyethyl cyclodextrinsand methyl cyclodextrins have already been used. These auxiliarysubstances were tested as filtration supporting agents to prevent gelformation.

[0015] No satisfactory solution of the problem could, however, be found.Only acidification with acetic acid showed partial success. Here, too,it was, however, always necessary to accept high filtration losses.

SUMMARY OF THE INVENTION

[0016] It was then surprisingly found that peptides having 3-15 aminoacids such as cetrorelix can be easily dissolved in 30% volume/volumeacetic acid. The solution is then diluted to a final concentration of 3%peptide, e.g., cetrorelix, with water for injection purposes andmannitol is added. Although it is stated in the literature that theterminal amide group hydrolyzes easily in acid medium, this was notfound in the case of cetrorelix. Solutions prepared according to thismethod caused no difficulties during filtration. The correct amounts ofactive substance were always found.

[0017] Thus, in accordance with the present invention, a peptide whichcontains 3-15 amino acids is dissolved in acetic acid to form a solutioncontaining 100-10,000 parts by weight of acetic acid for each part ofpeptide, the solution is transferred to water, and the resultingsolution is lyophilized.

[0018] The filtration speed of the acetic acid solution attains valuesthat ensure satisfactory production sequences. A general process forsterile lyophilization is described in pages 557-559 of Sucker, Fuchsand Speiser (Publishers) “Pharmazeutische Technologie” 2nd edition 1991,Thieme-Verlag, Stuttgart-New York. A further description of thelyphilization process used is given in German published specification(DOS) 37 35 614 (U.S. Pat. No. 5,204,335).

[0019] The lyophilizate is used in the treatment of female sterility.One therapeutic process has hitherto consisted in stimulating folliclematuration-using human menopause gonadotrophin and then triggeringovulation by administering human chorion gonadotrophin. The ovulationtriggered thereby occurred 32 hours later. The resulting ova areavailable for in vitro fertilization.

[0020] A disadvantage of this treatment with agonists is the fact thatup to 10 follicles mature during the stimulation phase. This elevatedfollicle maturation leads to hormone level peaks in the LH. These peaksresult in an early stage of follicle maturation and ovulation at anunpredicted point in time. This impaired ovulation occurs in about 25%of treated cases and is a disadvantage since the cycle that displaysdisturbed ovulation of this kind cannot be used for the collection ofova and the entire treatment has to be repeated about 1 month later.

[0021] Another disadvantage of the conventional simulation treatment andthe use of LHRH agonists in order to avoid premature LH-peaks is thelong treatment duration of 4 weeks which is needed to achievesatisfactory suppression. The agonists continue to display ahyperstimulation syndrome in 1-2% of cases in which the follicle cellshypertrophy. The risk of hyperstimulation is particularly great in thecase of polycystic ovaries. The hyperstimulation syndrome is a severeside effect which can lead to fatalities.

[0022] It has now been found that the antagonist cetrorelix displays thefollowing advantages in this treatment:

[0023] Treatment with cetrorelix over 5 days is sufficient to achievetotal suppression. The premature LH peaks cannot arise and the frequencyof hyperstimulation syndrome should be reduced. In addition, less HMG isused in the 2nd phase of therapy, the ovulation triggering phase. Thisgives this in-vitro fertilization treatment a not inconsiderable costadvantage. In-vitro fertilization is, for example, used when a tubeanomaly is present. To perform this treatment it is necessary toprecisely monitor the cycle and to establish the time of ovulation asprecisely as possible. This has hitherto only been achieved to a limitedextent since preovulatory LH increase often occurred too early due tosimulation with HMG/HCG, or was not maintained for a sufficiently longperiod. Avoidance of this premature increase is, however, of criticalimportance for the success of the treatment in order to preciselydetermine the time of fertilization. This reduces the physical andmental burden on the patient and makes optimum use of hospitallogistics. To achieve this objective with great reliability it isnecessary to suppress endogenous hormone production (LH-FSH, oestradiol)as completely as possible in order to simultaneously stimulate folliclematuration through administration of exogenous gonadotrophins (HMG/HCG)and to monitor the hormone status at any time. It is only when asufficiently large number of follicles have been achieved (4-6), havingapproximately the same degree of maturation, that ovulation is triggeredby administering an HCG bolus injection.

[0024] Use of an antagonist makes treatment substantially moresuccessful and safer for the patient.

[0025] Another area of use of the cetrorelix lyophilizate according tothe present invention is to protect the gonads in male patients. Malepatients are pre-treated with cetrorelix lyophilizate and the activityof the gonads is reinforced. As a result, other harmful noxious agents,such as radiation therapy or treatment with cytostatics, have no or onlya small possibility of affecting the sensitive tissue of the gonads.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0026] The following Examples illustrate the invention.

EXAMPLE 1

[0027] Approx. 1.5 liters of water for injection purposes are preparedin a suitable glass vessel. 210 g water for injection purposes areprepared in another glass vessel and 91.17 g acetic acid are added. Theamount of cetrorelix acetate calculated (1.62-1.695 g, depending on thecontent of the batch used) is dissolved in the prepared 30% acetic acidwith stirring. This solution is transferred to the glass vessel with 1.5liters of water for injection purposes, 82.2 g mannitol are added,dissolved and made up to 3039 g with water for injection purposes.In-process checks: pH value: 2.5-3.0 Density: 1.009-1.017 g/cm³ at 20°C. Refractive index: 1.227-1.340 at 440 nm and 20° C.

[0028] The solution is sterilized by filtration through an appropriatemembrane filter (pore size 0.2 μm) under aseptic conditions. 100 mlfirst runnings should be discarded. The filters should be sterilizedwith superheated steam before sterile filtration. Cetrorelixfreeze-dried solution should be protected from recontamination duringstorage.

[0029] The solution is immediately filled into colorless injectionbottles DIN 2R, hydrolytic class I under aseptic conditions and providedwith sterile freeze-drying stoppers. The nominal filling amount is 2.0ml=2.026 g.

[0030] The 2 ml injection bottles were rinsed in an injection bottlewashing machine, dried with hot air and sterilized. The cleaned,freeze-drying stoppers were autoclaved. The closed injection bottleswere transferred to a freeze-drying installation and frozen at a platetemperature of −40° C. Drying was carried out using a drying programwith a plate temperature of −40° C. rising to +20° C. The installationis then flooded with sterile nitrogen, the bottles are closed in theinstallation and the stoppers secured with crimped caps.

[0031] The injection bottles are checked visually for faulty closuresand outer faults. Faulty injection bottles are removed and destroyed.

[0032] Cetrorelix lyophilizate 1 mg is a white, solid, freeze-dried cakein a colorless 2 ml injection bottle which is closed with grayfreeze-drying stoppers and yellow flip-off crimped caps.

EXAMPLE 2

[0033] Nonapeptide (Bombesin-Antagonist)

[0034] 420 g water for injection purpose are prepared in a suitablevessel and 121.56 g acetic acid are added. The amount of the nonapeptide(about 3.783 g, depending on the content of the batch used) is dissolvedin the prepared 20% acetic acid and with stirring. 82., 2 niannitol areadded and dissolved. This solution is sterilized by filtration throughan appropriate membrane filter (pore size 0.2 μm) under asepticconditions. The same membrane filter is used for the water for injectionpurpose to make up the solution to 3064 g. The filters should besterilized with superheated steam. In-process checks: pH value: 2.5-3.0Density: 1.0213 -1.0225 g/cm³ at 20° C. Refractive Index: 1.335-1.345 at589 nm at 20° C.

[0035] The solution should be protected from recontamination duringstorage. The solution is filled in to sterile colorless injectionbottles DIN 2 R, hydrolytic class I under aseptic conditions andprovided with sterile freeze-drying stoppers. The nominal filling amountis 1.0 ml=1.022 g.

[0036] The 2 ml injection bottles were rinsed in an injection bottlewashing machine, dried with hot air and sterilized. The cleanedfreeze-drying stoppers were autoclaved. The injection bottles weretransferred to a freeze-drying installation and frozen at a platetemperature of −40° C.

[0037] Drying was carried out using a drying programme with a platetemperature of −40° C. rising to +20° C. The installation is thenflooded with sterile nitrogen, the bottles are closed in theinstallation and the stoppers are sealed with crimped caps.

[0038] The injection bottles are checked visually for faulty closuresand outer faults. Faulty injection bottles are removed and destroyed.

[0039] The lyophilisate of the nonapeptide (1 mg) is a white, solid,freeze-dried cake in a colorless 2 ml injection bottle which is closedwith grey freeze-drying stoppers and flip-off crimped caps.

EXAMPLE 3

[0040] Tripeptide (Protirelin)

[0041] 143.5 g water for injection purpose are prepared in a suitablevessel and 61.5 g acetic acid are added. The amount of the Protirelinacetate calculated (equivalent to 800 mg of the peptide base) isdissolved with stirring. This solution is transferred to another vesselwith approximately 1 l water for injection purpose. 109.6 g mannitol areadded, dissolved and made up to 2048 g with water for injectionpurposes. In-process checks: pH value: 2.5-3.0 Density: 1.0232-1.0252g/cm³ at 20° C. Refractive Index: 1.334-1.344 at 589 nm at 20° C.

[0042] The solution is filled in to sterile colorless injection bottlesDIN 2 R, hydrolytic class I under aseptic conditions and provided withsterile freeze-drying stoppers. The nominal filling amount is 1.0 ml1.024 g.

[0043] The 2 ml injection bottles were rinsed in an injection bottlewashing machine, dried with hot air and sterilized. The cleanedfreeze-drying stoppers were autoclaved. The injection bottles weretransferred to a freeze-drying installation and frozen at a platetemperature of −40° C.

[0044] Drying was carried out using a drying programme with a platetemperature of −40° C. rising to +20° C. The installation is thenflooded with sterile nitrogen, the bottles are closed in theinstallation and the stoppers are sealed with crimped caps.

[0045] The injection bottles are checked visually for faulty closuresand outer faults. Faulty injection bottles are removed and destroyed.

[0046] The Protireline lyophilizate (0.4 mg) is a white, solid,freeze-dried cake in a colorless 2 ml injection bottle which is closedwith grey freeze-drying stoppers and flip-off crimped caps.

EXAMPLE 4

[0047] Tetradecapeptide (Somatostatin)

[0048] 245 g water for injection purpose are prepared in a suitablevessel and 61.5 g acetic acid are added. The amount of somatostatineacetate calculated (0.52-0.66 g, dependent on the content of the batchused) is dissolved with stirring. This solution is transferred toanother vessel with approximately 1 l water for injection purpose. 109.6g mannitol are added, dissolved and made up to 2049 g with water forinjection purposes. In-process checks: pH value: 2.5-3.0 Density:1.0235-1.0255 g/cm³ at 20° C. Refractive Index: 1.336-1.348 at 589 nm at20° C.

[0049] The solution is filled in to sterile colorless injection bottlesDIN 2 R, hydrolytic class I under aseptic conditions and provided withsterile freeze-drying stoppers. The nominal filling amount is 1.0 ml1.024 g.

[0050] The 2 ml injection bottles were rinsed in an injection bottlewashing machine, dried with hot air and sterilized. The cleanedfreeze-drying stoppers were autoclaved. The injection bottles weretransferred to a freeze-drying installation and frozen at a platetemperature of −40° C.

[0051] Drying was carried out using a drying programme with a platetemperature of −40° C. rising to +20° C. The installation is thenflooded with sterile nitrogen, the bottles are closed in theinstallation and the stoppers are sealed with crimped caps.

[0052] The injection bottles are checked visually for faulty closuresand outer faults. Faulty injection bottles are removed and destroyed.

[0053] The lyophilizate (0.25 mg somatostatine acetate) is a white,solid, freeze-dried cake in a colorless 2 ml injection bottle which isclosed with grey freeze-drying stoppers and flip-off crimped caps.

What is claimed is:
 1. A method of making a lyophilizate of a peptide which contains 3-15 amino acids, said method comprising dissolving the peptide in acetic acid to form a solution containing 100-10,000 parts by weight of acetic acid for each part of peptide, transferring the solution to water, and lyophilizing the resulting solution.
 2. A method as set forth in claim 1 in which at least one bulking agent is dissolved in the acetic acid with the peptide.
 3. A method as set forth in claim 2 in which the bulking agent is mannitol.
 4. A method as set forth in claim 1 in which the peptide is selected from the group consisting of Bombesin-Antagonist, Protirelin, Somatostatin and Cetrorelix.
 5. A method as set forth in claim 1 in which the peptide is selected from the group consisting of Bombesin-Antagonist, Protirel and Somatostatin.
 6. A method set forth in claim 1 in which the peptide is cetrorelix.
 7. A pharmaceutical composition containing a peptide produce by the method of any one of claims 1-5.
 8. A pharmaceutical composition containing a tide produced by the method of claim
 6. 9. A method of treating female infertility which comprises administering a effective of the pharmaceutical composition of claim
 8. 10. A method of providing male gonad protection which comprises administering to a male an effective amount of the pharmaceutical composition of claim
 8. 